1. Field of the Invention
The present invention is directed toward a cell culture assembly used in the biomedical science field of tissue engineering and, more specifically, to a cell culture assembly through which fluid may flow for applying deformations to cells that include fluid-induced stress or substrate-induced strain to cultured cells.
2. Prior Art
In the human body, many cells are constantly subjected to stress from fluid flow. Fluid flow in the body includes blood flow through the vasculature, lymph in the lymphatics, cerebrospinal fluid flow, any secretion in ducts, and also the movement of interstitial fluid in the matrix between and among cells in any tissue. Stressing cells in culture simulates the in vivo environment, causing dramatic morphologic changes and biomechanical responses in the cells. There are both long and short-term changes that occur when cells are stressed in culture, such as alterations in the rate and amount of protein expression and secretion, the rate of cell division and alignment, changes in energy metabolism, changes in rates of macromolecular synthesis or degradation, and other changes in biochemistry and bioenergetics. Prior devices have been developed for applying substrate deformation on cells and applying fluid-induced shear stress by subjecting the cells to fluid flow. However, none of these devices have allowed for alternating or simultaneous application of both types of mechanical loading of cells in vitro and for quantitation of the applied stresses and strains.
A need remains for a cell culture assembly in which cells may be cultured and subjected to fluid-induced shear stress which is precisely controlled.
Accordingly, I have developed a cell culture assembly including a body having a flow surface extending across an upper surface of the body. The top surface of the body may also be used as a flow surface on which cells may be cultured. Moreover, a flexible membrane may be clamped by the body and also be used as a flow surface on which cells may be cultured. This rubber membrane may also be deformed by vacuum so this cell receives substrate tension in unconstrained distension may be deformed by stretching across a planar faced post so that the flexible substrate is deformed equibiaxially. Positive pressure may also be applied to deform the flexible membrane upward to apply a compressive deformation to overlying cells cultured on the top member. Both fluid stress and substrate strain may also be delivered simultaneously as often occurs in blood vessels or in other tissues.
The body further defines a passageway in fluid communication with the flow surface and a cover member covering the flow surface. The flow surface of the body and the cover member thereby define a flow chamber through which fluid may flow. A cell culture surface is positioned on the flow surface or on the cover or both. Cells cultured on the cell culture surface are subject to shear stress when fluid flows through the passageway and the flow chamber.
In one embodiment of the invention, the body has an upper surface defining a first opening therethrough. The assembly further includes a base attached to the body and a cell culture membrane fixed between the base and the body whereby the membrane covers the first opening, such that when fluid flows through the body passageway, the fluid passes across the membrane thereby inducing shear stress on cells growing on the membrane. The body passageway includes a pair of bores defined in the body on opposing sides of the first opening, wherein each bore extends between a side of the body and the upper surface. The upper surface defines a pair of second openings, preferably in the form of slits, on opposing sides of the first opening and each second opening is in fluid communication with one of the bores. A gasket is positioned on the body upper surface and surrounds the first opening and the second openings. The gasket is configured to retain fluid flowing out of one of the second openings and into the other second opening. A port is defined in the body for connection to a pressure supply. The body upper surface further defines an annular channel in fluid communication with the port. The gasket overlies the channel and the cover overlies the gasket. The gasket defines a plurality of holes which overlie the annular channel such that the cover seats on the gasket when negative pressure is applied to the port. Alternatively, the upper surface may be clamped by overlying pressure to the gasket and body by conventional assemblies such as a plate and fasteners.
The base comprises an annular member defining a chamber and having a wall with a top surface on which the membrane is seated. An insert is received within the chamber. The insert includes a support member with a support surface for supporting a portion of the membrane. The wall of the base defines an aperture and the insert defines an insert passageway extending between a side of the insert and the insert support surface where the insert passageway is in fluid communication with the aperture of the base wall. When negative pressure is applied to the chamber through the aperture, the membrane is urged against the insert support surface. Preferably, the insert includes a post spaced apart from the support member thereby defining an annular gap between the post and the support member. An opening defined in the support member is in fluid communication with the gap. Preferably, an upper surface of the post is lower than the support surface and an upper surface of the portion of the membrane supported by the support surface is in a plane of the upper surface of the body.
In another embodiment of the invention, the body includes a flow member and a pair of end members attached to opposing ends of the flow member, where the opposing ends of the flow member each define a recess, and where each flow surface extends between the recesses in the ends of the flow member. In this arrangement, the openings are defined in the end members and are in fluid communication with the recesses. An annular channel is defined in an upper surface of the body and surrounds the flow surface. The body defines a port in fluid communication with the channel whereby when negative pressure is applied to the port, the cover is urged toward the body. A gasket defining an opening aligned with the flow surface and defining a plurality of holes therethrough is positioned between the body and the cover. The gasket opening overlies the flow surface and the holes overlie the channel, such that when negative pressure is applied to the port, the cover sealingly seats on the gasket and the gasket sealingly seats on the body. The flow chamber is defined by the gasket, the cover and the recess. The body may include a plurality of flow surfaces with the channel surrounding each flow surface defined in the body, and the gasket defining a plurality of openings each overlying a flow surface. Each end of the body then defines a plurality of openings aligned with each of the flow surfaces.
Another embodiment of the invention includes a body, a top, and a bottom in fluid communication with each other for the steady flow of fluid therethrough. The body has a plurality of flow shafts extending therethrough. The flow shafts are preferably substantially parallel to each other. A plurality of slides is inserted into the plurality of flow shafts. Cells are cultured on the plurality of slides. The top is in fluid communication with the plurality of flow shafts. The bottom is also in fluid communication with the plurality of flow shafts. Fluid is delivered, via the top and/or the bottom, to the flow shafts to simulate the stresses on the cells in blood vessels or other tissues caused by fluid flow.
The body of the assembly may include O-ring grooves surrounding each end of the plurality of flow shafts. O-rings are placed in the O-ring grooves to ensure a leakproof seal between the body and the top and bottom when assembled. Alternatively, O-ring grooves may be located on the top or bottom to surround each end of the plurality of flow shafts when the assembly is assembled.